1. Field of the Invention
The present invention relates to antianemia ingredients extracted from traditional Chinese herbal medicine Trichosanthes, which comprise a single compound with the activity of increasing globin gene expression. This invention also relates to methods of preparing the ingredients and purifying the compound, and uses thereof.
2. Description of the Related Art
The hereditary hemoglobinopathies such as sickle cell anemia and thalassemias are among the most prevalent serious genetic disorders affecting human populations and represent a major health burden worldwide. Even nowadays, blood transfusions remain the major means to ameliorate the clinical anemia syndromes although they are only of temporary benefit. Hypertransfusion induced iron overload requires effective iron chelating therapy to improve growth and prolong survival in patients. Bone marrow transplantation can cure the disease but has not been widely performed because of risk, expense, the need for an HLA compatible donor and poor acceptance by families and patients.
Recently, the search for treatment aimed at reduction of globin chain imbalance in patients with thalassemia has focused on the pharmacologic manipulation of fetal hemoglobin (α2γ2; HbF). The switch from fetal hemoglobin to adult hemoglobin (α2β2; HbA) usually proceeds within six months after parturition, which unfortunately also proceeds on schedule in patients with hemoglobinopathies. However, in the majority of these individuals, the upstream γ globin genes are intact and fully functional, and if these could be reactivated, functional hemoglobin synthesis could be maintained during adulthood, ameliorating the severity of the disease (Atweh, G. F., and D. Loukopoulos, 2001, Pharmacological induction of fetal hemoglobin in sickle cell disease and beta-thalassemia, Semin Hematol, 38: 367-73). This is suggested by observations of the mild phenotype of individuals with co-inheritance of homozygous β-thalassemia and hereditary persistence of fetal hemoglobin (HPFH), and by those patients with homozygous β0-thalassemia who synthesize no adult hemoglobin, but in whom a reduced requirement for transfusions is observed in the presence of increased concentrations of fetal hemoglobin.
The first group of compounds discovered having HbF reactivation activity are cytotoxic drugs. 5-azacytidine was initially found, in experimental animals, impressing cellular control of fetal hemoglobin in the adult (DeSimone, J., P. Heller, L. Hall and D. Zwiers, 1982, 5-Azacytidine stimulates fetal hemoglobin synthesis in anemic baboons, Proc Natl Acad Sci USA, 79: 4428-31). Later baboons treated with cytotoxic doses of arabinosylcytosine (ara-C) responded with striking elevations of F-reticulocytes (Papayannopoulou, T., A. Torrealba de Ron, R. Veith, G. Knitter and G. Stamatoyannopoulos, 1984, Arabinosylcytosine induces fetal hemoglobin in baboons by perturbing erythroid cell differentiation kinetics, Science, 224: 617-9). Induction of γ-globin also occurred in monkeys or baboons treated with hydroxyurea (Letvin, N. L., D. C. Linch, G. P. Beardsley, K. W. McIntyre and D. G. Nathan, 1984, Augmentation of fetal-hemoglobin production in anemic monkeys by hydroxyurea, N Engl J Med, 310: 869-73). Vinblastine, an M stage-specific agent that arrests cells in mitosis, also produces perturbations of erythropoiesis and stimulates HbF synthesis in baboons (Veith, R., T. Papayannopoulou, S. Kurachi and G. Stamatoyannopoulos, 1985, Treatment of baboon with vinblastine: insights into the mechanisms of pharmacologic stimulation of Hb F in the adult, Blood, 66: 456-9). Following these studies, hydroxyurea was used for induction of HbF in humans and later became the first and only drugs approved by the Food and Drug Administration (FDA) for the treatment of hemoglobinopathies. However, the pharmacologic induction of HbF through the mechanism of late progenitor cell cytotoxicity seems to reach a dead end in drug discovery. It is unlikely that a cytotoxic compound better than hydroxyurea could be found.
The second group of compounds owning to HbF reactivation activity was short chain fatty acids. Initially, the seminal observation led to the discovery of γ-aminobutyric acid, which is acting as a fetal hemoglobin inducer (Perrine, S. P., B. A. Miller, M. F. Greene, R. A. Cohen, N. Cook et al., 1987, Butryic acid analogues augment gamma globin gene expression in neonatal erythroid progenitors, Biochem Biophys Res Commun, 148: 694-700). Subsequent studies showed that butyrate stimulated globin production in adult baboons (Constantoulakis, P., T. Papayannopoulou and G. Stamatoyannopoulos, 1988, alpha-Amino-N-butyric acid stimulates fetal hemoglobin in the adult, Blood, 72: 1961-7), and it induced y globin in erythroid progenitors in adult animals or patients with sickle cell anemia (Perrine, S. P., B. A. Miller, D. V. Faller, R. A. Cohen, E. P. Vichinsky et al., 1989, Sodium butyrate enhances fetal globin gene expression in erythroid progenitors of patients with Hb SS and beta thalassemia, Blood, 74: 454-9). Derivatives of short chain fatty acids such as phenylbutyrate (Dover, G. J., S. Brusilow and S. Charache, 1994, Induction of fetal hemoglobin production in subject,s with sickle cell anemia by oral sodium phenylbutyrate, Blood, 84: 339-43) and valproic acid (Liakopoulou, E., C. A. Blau, Q. Li, B. Josephson, J. A. Wolf et al., 1995, Stimulation of fetal hemoglobin production by short chain fatty acids, Blood, 86: 3227-35) also induce HbF in vivo. Since there exist a very large number of short chain fatty acid analogs or derivatives that are potential inducers of HbF, there are ample opportunities for discovering HbF inducers that are more potent than butyrate. Phenylacetic and phenylalkyl acids (Torkelson, S., B. White, D. V. Faller, K. Phipps, C. Pantazis et al., 1996, Erythroid progenitor proliferation is stimulated by phenoxyacetic and phenylalkyl acids, Blood Cells Mol Dis, 22: 150-8), which were discovered during following studies, belonged to such examples. Presently, the use of butyrate or its analogs in sickle cell anemia and β-thalassemia remains experimental and cannot be recommended for treatment outside of clinical trials.
Clinical trials aimed at reactivation of fetal hemoglobin synthesis in sickle cell anemia and β-thalassemia have included short term and long term administration of 5-azacytidine, hydroxyurea, recombinant human erythropoietin, and butyric acid analogs, as well as combinations of these agents. However, varying drawbacks contraindicate the long term use of such agents or therapies. For example, although the hydroxyurea stimulates fetal hemoglobin production and clinically reduces sickling crisis, it is potentially limited by myelotoxicity and the risk of carcinogenesis. Potential long term carcinogenicity also exists in 5-azacytidine-based therapies. Feasible clinical treatments for these diseases remain scarce. Erythropoietin-based therapies have not proved consistent among a range of patient populations. The short half-lives of butyric acid in vivo have been viewed as a potential obstacle in clinical settings.
Still, notable efforts have been made in discovery and developing new drugs for the treatment of sickle cell anemia and β-thalassemia. In the past decades, some progress has also been made in the pharmacological management of sickle cell anemia and thalassemia.
WO 9,712,855 (Tung Roger D.) describes butyrate prodrugs derived from lactic acid for increasing gamma globin and fetal hemoglobin in a patient. The compounds disclosed in the application are particularly effective in treating beta-hemoglobinopathies, including sickle cell syndromes and beta-thalassemia syndromes.
U.S. Pat. No. 6,372,213 (Um, et al.) provides a method of treatment of sickle cell anemia or thalassemia with protein C. The patent provides a needed therapy for potentially serious and debilitating disorders while avoiding complications such as bleeding tendency, toxicity and general side effects of currently available anti-coagulant agents.
U.S. Pat. No. 6,312,707 (Markov, et al.) describes fructose-1,6-diphosphate (FDP) has been shown, in double-blinded controlled clinical trials on patients with sickle cell anemia, to substantially reduce the pain suffered by such patients during the recurrent ischemic crises that are caused by red blood cell sickling.
U.S. Pat. No. 6,231,880 (Perrine) describes a number of compositions for pulsed administration to treat human blood disorders such as sickle cell anemia or thalassemia. The compositions contain chemical compounds that stimulate the expression of fetal hemoglobin and stimulate the proliferation of red blood cells, white blood cells and platelets in patients and ex vivo for reconstitution of hematopoiesis in vivo.
U.S. Pat. No. 6,028,103 (Brugnara, et al.) discloses that triaryl methane compounds or analogues are useful as efficacious drugs in the treatment of sickle cell disease and diseases characterized by unwanted or abnormal cell proliferation. The compounds inhibit mammalian cell proliferation, inhibit the Gardos channel of erythrocytes, reduce sickle erythrocyte dehydration and delay the occurrence of erythrocyte sickling or deformation.
U.S. Pat. No. 5,945,407 (Bemis, et al.) describes uses of butyrate esters of threitol in pharmaceutically increasing fetal hemoglobin and gamma globin in a patient, and particularly in treating beta-hemoglobinopathies, such as sickle cell syndromes and beta-thalassemia syndromes.
U.S. Pat. No. 5,753,632 (Schmidt, et al.) describes the use of colloidal silica for the treatment of sickle-cell anemia, malaria and exogenously induced leucopenias, which leads to a significant improvement in the condition of the patients.
U.S. Pat. No. 5,665,392 (Kumar, et al.) describes a pharmaceutical formulation useful for treating patients suffering from thalassemia, which comprises powder of Anemonin Pretensis in an amount in the range of 0.02 to 0.12 wt % of the formulation, quinine sulphate in an amount in the range of 0.0005 to 0.003 wt % of the formulation, distilled or demineralised water in an amount in the range of 0 to 40 wt % of the formulation and, ethanol in an amount in the range of 99.88 to 60 wt % of the formulation; and a process for preparing the formulation by mixing the above ingredients.
U.S. Pat. No. 5,447,720 (Fadulu) describes a composition extracted from alfalfa and other certain plant materials for the treatment of hemoglobinopathies. The plant material is first extracted with 1,1,1-trichloroethane and a hydroxide base, followed by extraction with hexane. The polar acidic compounds present in alfalfa and other plant materials selectively dissolve in the hexane phase and exhibit good antisickling activity in vitro. Further, these active compounds which comprise the inventive extract are effective in vivo by significantly alleviating the many clinical manifestations of sickle cell anemia and thalassemia patients.
EP 0,627,220 (Hayhurst Walter) describes pharmaceutical compositions containing butyric acid derivatives, particularly isobutyramide, are advantageously indicated for the therapy of thalassemia when compared with known formulations.
EP 0,617,966 (Perrine Susan P.) describes a method for inhibiting the gamma-globin to beta-globin switching in subjects afflicted with beta-globin disorders. It ameliorates the clinical symptoms of sickle cell disease or beta-thalassemia by introducing activin or inhibin into the subject prior to natural completion of the switching process.
Although these efforts lead advances in developing new drugs for the treatment of hemoglobinopathies, most of them are still under further investigation, as still far from practical use. It is apparent that the present treatment of the sickle cell anemia and β-thalassemia is not ideal. Limited effectiveness, risks, expenses, and difficulties in compliance characterize most of the therapies currently available. Accordingly, it is urgent to develop alternatives having advantages over the prior art, and avoiding the disadvantages of those disclosed in the prior art, while providing effective therapy for those target diseases.